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PubMed2000

Virtual electrode polarization in the far field: implications for external defibrillation.

We recently suggested that failure of implantable defibrillation therapy may be explained by the virtual electrode-induced phase singularity mechanism. The goal of this study was to identify possible mechanisms of vulnerability and defibrillation by externally applied shocks in vitro. We used bidomain simulations of realistic rabbit heart fibrous geometry to predict the passive polarization throughout the heart induced by external shocks. We also used optical mapping to assess anterior epicardium electrical activity during shocks in Langendorff-perfused rabbit hearts (n = 7). Monophasic shocks of either polarity (10-260 V, 8 ms, 150 microF) were applied during the T wave from a pair of mesh electrodes. Postshock epicardial virtual electrode polarization was observed after all 162 applied shocks, with positive polarization facing the cathode and negative polarization facing the anode, as predicted by the bidomain simulations. During arrhythmogenesis, a new wave front was induced at the boundary between the two regions near the apex but not at the base. It spread across the negatively polarized area toward the base of the heart and reentered on the other side while simultaneously spreading into the depth of the wall. Thus a scroll wave with a ribbon-shaped filament was formed during external shock-induced arrhythmia. Fluorescent imaging and passive bidomain simulations demonstrated that virtual electrode polarization-induced scroll waves underlie mechanisms of shock-induced vulnerability and failure of external defibrillation.

Animals↗
PubMed2005

Mechanoelectrical excitation by fluid jets in monolayers of cultured cardiac myocytes.

Although the prevailing view of mechanoelectric feedback (MEF) in the heart is in terms of longitudinal cell stretch, other mechanical forces are considerable during the cardiac cycle, including intramyocardial pressure and shear stress. Their contribution to MEF is largely unknown. In this study, mechanical stimuli in the form of localized fluid jet pulses were applied to neonatal rat ventricular cells cultured as confluent monolayers. Such pulses result in pressure and shear stresses (but not longitudinal stretch) in the monolayer at the point of impingement. The goal was to determine whether these mechanical stimuli can trigger excitation, initiate a propagated wave, and induce reentry. Cells were stained with the voltage-sensitive dye RH237, and multi-site optical mapping was used to record the spread of electrical activity in isotropic and anisotropic monolayers. Pulses (10 ms) with velocities ranging from 0.3 to 1.8 m/s were applied from a 0.4-mm diameter nozzle located 1 mm above the cell monolayer. Fluid jet pulses resulted in circular wavefronts that propagated radially from the stimulus site. The likelihood for mechanical stimulation was quantified as an average stimulus success rate (ASSR). ASSR increased with jet amplitude and time waited between stimuli and decreased with the application of gadolinium and streptomycin, blockers of stretch-activated channels, but not with nifedipine, a blocker of the L-type Ca channel. Absence of cellular injury was confirmed by smooth propagation maps and propidium iodide stains. In rare instances, the mechanical pulse resulted in the induction of reentrant activity. We conclude that mechanical stimuli other than stretch can evoke action potentials, propagated activity, and reentrant arrhythmia in two-dimensional sheets of cardiac cells.

Animals↗
PubMed2006

Gap junctions and propagation of the cardiac action potential.

Pacemaker cells in the heart generate periodic electrical signals that are conducted to the working myocardium via the specialized conduction system. Effective cell-to-cell communication is critical for rapid, uniform conduction of cardiac action potentials-- a prerequisite for effective, synchronized cardiac contraction. Local circuit currents form the basis of the depolarization wave front in the working myocardium. These currents flow from cell to cell via gap junction channels. In this chapter, we trace the path of the action potential from its generation in the sinus node to propagation through the working myocardium, with a detailed discussion of the role of gap junctions. First, we review the transmembrane ionic currents and the basic principles of conduction of the action potential to the working myocardium via the specialized tissues of the heart. Next, we consider the relative contribution of cell geometry, size, and gap junction conductance. These factors are examined in terms of their source-to-sink relationships. Lastly, we will discuss new insights into the importance of gap junctions in cardiac conduction in health and disease which have been gained from high resolution optical mapping in connexin-deficient mice.

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PubMed2002

Mice display sex differences in halothane-induced polymorphic ventricular tachycardia.

BACKGROUND: Molecularly engineered mice are extensively used as models of cardiovascular diseases, yet little is known about sex differences in the electrophysiology of mouse hearts. METHODS AND RESULTS: This study investigated the influence of sex on drug-induced polymorphic ventricular tachycardia (PVT) in Langendorff-perfused male and female mice hearts (n=54) by injecting a bolus of halothane (1.75 mmol/L) in the perfusate while recording ECGs or optical action potentials (APs). There were no statistically significant differences between male and female hearts (n=54) with respect to mean RR (193+/-5 ms), PR (47+/-1 ms), QT intervals (101+/-3 ms), optical AP durations (APD(75)=23.11+/-4.2 ms), dispersion of refractory periods, and conduction velocities (n=5 male and 5 female). Halothane induced PVTs lasting a mean duration of 90 seconds; in female hearts, 55% of PVTs lasted longer than the median, whereas in male hearts 17% exceeded the mean (P<0.05). The total duration of PVTs exposed a marked sex difference, 378+/-144 seconds in female versus 27+/-10 seconds in male hearts (P<0.05). In optically mapped male hearts, halothane reduced APD(75) (17.61+/-1.6 ms) and then elicited VTs (n=6 of 6), but in female hearts, halothane elicited PVTs (n=1 of 6) or arrested the hearts (n=5 of 6). Except for KCNE1, Northern blots (KCNQ1, MERG, Kv1.5, connexins 40 and 43, TREK1, and TASK1) did not detect sex differences. CONCLUSIONS: This mouse model reveals sex difference in response to a pharmacological challenge yet does not display sex differences in standard electrophysiological parameters. Differences in KCNE1 may contribute to sex differences uncovered by halothane.

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PubMed2003

Hysteresis effect implicates calcium cycling as a mechanism of repolarization alternans.

BACKGROUND: T-wave alternans is due to alternation of membrane repolarization at the cellular level and is a risk factor for sudden cardiac death. Recently, a hysteresis effect has been reported in patients whereby T-wave alternans, once induced by rapid heart rate, persists even when heart rate is subsequently slowed. We hypothesized that alternans hysteresis is an intrinsic property of cardiac myocytes, directly related to an underlying mechanism for repolarization alternans that involves intracellular calcium cycling. METHODS AND RESULTS: Stepwise pacing was used to induce alternans in Langendorff-perfused guinea pig hearts from which optical action potentials were recorded simultaneously at 256 ventricular sites with voltage-sensitive dyes and in whole-cell patch-clamped cardiac myocytes treated with or without BAPTA-AM (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid tetrakis [acetoxymethyl ester]). Alternans hysteresis was observed in every isolated heart: threshold heart rate for alternans was 280+/-12 bpm, but during subsequent deceleration of pacing, alternans persisted to significantly slower heart rates (238+/-5 bpm, P<0.05). Optical mapping showed that this effect also applied to the threshold for spatially discordant alternans (313+/-2.2 bpm during acceleration versus 250+/-6.6 bpm during deceleration, P<0.05). Alternans hysteresis was also observed in isolated cardiac myocytes. Moreover, calcium chelation by BAPTA-AM raised the threshold for alternans and inhibited hysteresis in a dose-dependent manner with no effect on baseline action potential duration. CONCLUSIONS: Alternans hysteresis is an intrinsic property of cardiac myocytes that can lead to persistence of arrhythmogenic discordant alternans even after heart rate is slowed. These results also support an important underlying role of calcium cycling in the mechanism of alternans.

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PubMed2003

Pharmacological modulation of cardiac gap junctions to enhance cardiac conduction: evidence supporting a novel target for antiarrhythmic therapy.

BACKGROUND: Disease-induced alterations of cardiac gap junctions lead to intercellular uncoupling, which is an important mechanism of arrhythmogenesis. Therefore, drugs that selectively open gap junctions potentially offer a novel strategy for antiarrhythmic therapy. Because the peptide ZP123 was found to increase conductance between paired myocytes, we hypothesized that ZP123 would suppress acidosis-induced gap junction closure in the intact heart. METHODS AND RESULTS: High-resolution optical mapping was used to measure conduction velocity (CV) and action potential duration from ventricular epicardium of Langendorff-perfused guinea pig hearts at baseline (pH 7.4) and during 45 minutes of perfusion with acidotic (pH 6.0) Tyrode's solution with (n=8) and without (control, n=7) ZP123 (80 nmol/L). Acidosis produced conduction slowing transverse (29.1+/-0.1 to 16.8+/-0.2 cm/s, P<0.0001) and longitudinal (47.2+/-2.4 to 33.2+/-4.8 cm/s, P<0.0001) to cardiac fibers. Importantly, ZP123 inhibited conduction slowing during acidosis by approximately 60%. The peak effect of ZP123 was achieved after 16 minutes of acidosis, consistent with inhibition of uncoupling. ZP123 did not affect Na+ current in isolated myocytes, additionally affirming that preservation of CV was attributable to the compound's action on gap junctions. ZP123 had no effect on CV in the absence of acidosis, suggesting that drug activity targets gap junctions under metabolic stress. Action potential duration heterogeneity was significantly reduced by ZP123 (6.7+/-0.8 ms) compared with controls (9.7+/-3.1 ms, P<0.05), presumably by enhancing cell-to-cell coupling. CONCLUSIONS: These data suggest that ZP123 significantly attenuates gap junction closure during acidosis. Preservation of intercellular coupling diminished CV slowing and heterogeneous repolarization, eliminating arrhythmogenic substrates.

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PubMed2004

Transcription enhancer factor-1-related factor-transgenic mice develop cardiac conduction defects associated with altered connexin phosphorylation.

BACKGROUND: Conduction system defects and slowed ventricular conduction are common in patients with systolic dysfunction and contribute to arrhythmias and sudden death. In animal models of heart failure, cardiac alpha1-adrenergic signaling is constitutively activated. Here, we report the effects of constitutive activation of alpha1-adrenergic signaling on connexin phosphorylation and cardiac conduction. METHODS AND RESULTS: Transgenic mice were generated with cardiac-specific overexpression of the transcription factor RTEF-1 (transcription enhancer factor-1-related factor), which mediates alpha1-adrenergic signaling in cardiac myocytes. Surface and intracardiac ECGs revealed prolongation of the PR, QRS, and AH intervals and the appearance of progressive atrial arrhythmias in RTEF-1 mice. Optical mapping using voltage-sensitive dye revealed slower conduction velocities across the atrial and ventricular myocardium. Intercellular dye transfer between RTEF-1 transgenic cardiac myocytes confirmed impaired conduction at the cellular level. Conduction defects were correlated with dephosphorylation of connexin40 and connexin43 and upregulation of protein phosphatase 1beta (PP1beta). Overexpression of PP1beta in HeLa cells dephosphorylated cardiac connexin. Confocal microscopy revealed increased levels of dephosphorylated connexin43 at the cardiac gap junctions in RTEF-1 mice, suggesting that defective conduction is a result of impaired gap-junction conductance rather than assembly. CONCLUSIONS: Constitutive activation of alpha1-adrenergic signaling through the RTEF-1 transcription factor results in chronic elevation of PP1beta expression and connexin dephosphorylation. This mechanism may underlie some defects in cardiac conduction.

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PubMed2004

Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers.

BACKGROUND: Human embryonic stem cells (hESCs) derived from blastocysts can propagate indefinitely in culture while maintaining pluripotency, including the ability to differentiate into cardiomyocytes (CMs); therefore, hESCs may provide an unlimited source of human CMs for cell-based therapies. Although CMs can be derived from hESCs ex vivo, it remains uncertain whether a functional syncytium can be formed between donor and recipient cells after engraftment. METHODS AND RESULTS: Using a combination of electrophysiological and imaging techniques, here we demonstrate that electrically active, donor CMs derived from hESCs that had been stably genetically engineered by a recombinant lentivirus can functionally integrate with otherwise-quiescent, recipient, ventricular CMs to induce rhythmic electrical and contractile activities in vitro. The integrated syncytium was responsive to the beta-adrenergic agonist isoproterenol as well as to other pharmacological agents such as lidocaine and ZD7288. Similarly, a functional hESC-derived pacemaker could be implanted in the left ventricle in vivo. Detailed optical mapping of the epicardial surface of guinea pig hearts transplanted with hESC-derived CMs confirmed the successful spread of membrane depolarization from the site of injection to the surrounding myocardium. CONCLUSIONS: We conclude that electrically active, hESC-derived CMs are capable of actively pacing quiescent, recipient, ventricular CMs in vitro and ventricular myocardium in vivo. Our results may lead to an alternative or a supplemental method for correcting defects in cardiac impulse generation, such as cell-based pacemakers.

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PubMed2000

Mechanism of ventricular defibrillation. The role of tissue geometry in the changes in transmembrane potential in patterned myocyte cultures.

BACKGROUND: The geometry of the myocardium may influence changes in transmembrane potential (DeltaVm) during defibrillation. To test this hypothesis, specific nonlinear structures (bifurcations, expansions, and curved strands or "bends") were created in patterned cultures of neonatal rat myocytes. METHODS AND RESULTS: Extracellular field stimuli (EFS; 7 to 11 V/cm field strength) were applied parallel to the strands. Changes in Vm were measured with microscopic resolution using optical mapping techniques. In bifurcations, EFS produced 2 DeltaVm maxima (so-called secondary sources) at the shoulder of each limb that were separated by a decrease of either hyperpolarization or depolarization at the insertion of the stem strand. In expansions, EFS produced a significant decrease in DeltaVm at the insertion site of the expansion compared with the DeltaVm maxima measured at the lateral borders. In 50% of experiments, tertiary sources of opposite polarity appeared in the strand due to local electrotonic currents. New action potentials were propagated from the sites of DeltaVm maxima located at the lateral borders of the expansions. In bends, the strand oriented in parallel to the field dominated electrotonically and partially cancelled the sources produced by the perpendicular segment. CONCLUSIONS: In electrically well-coupled nonlinear structures, EFS produced changes in Vm at resistive boundaries that were determined by the electrotonic interaction between sources of different, direction-dependent strength. In addition, the interaction between localized secondary sources at nonlinear boundaries generated local current circuits, which gave rise to further changes in Vm (tertiary sources).

Animals↗
PubMed1991

Repolarization inhomogeneities in ventricular myocardium change dynamically with abrupt cycle length shortening.

BACKGROUND: In single heart cells, abrupt changes in stimulation rate elicit complex alterations in repolarization. The effects of rate change on dispersion of repolarization, however, have not been well characterized. METHODS AND RESULTS: To determine the effects of abrupt cycle length (CL) shortening on spatial inhomogeneity of repolarization in a syncytium of ventricular cells, 124 action potentials were simultaneously recorded from Langendorff-perfused guinea pig hearts using high-resolution optical mapping with voltage-sensitive dye. The distribution of ventricular action potential durations (APDs) mapped during each cardiac cycle was used to calculate mean APD and repolarization dispersion index (DI), defined as the variance of the distribution. After abruptly shortening CL from 500 to 300 msec, mean APD declined exponentially in normoxic controls (by 23 +/- 3 msec, p less than 0.0001). This response was characterized by beat-to-beat oscillations of APD that were synchronized at all ventricular sites. After 30 minutes of hypoxia, mean APD decreased from 175.0 +/- 13.3 to 76 +/- 25.7 msec. However, during hypoxia, abrupt CL shortening lowered mean APD by only an additional 6 +/- 6 msec, and APD oscillations were no longer synchronized throughout the ventricle. In controls, beat-to-beat DI decreased significantly (-51.0 +/- 6.8%, p less than 0.01) by the sixth post-CL shortening beat and then recovered (by 15-20 beats). In contrast, DI failed to decrease during hypoxia (+7.1 +/- 23%). Two mechanisms for the transient decline of DI in controls were identified: synchronous APD oscillations and transient diminution of the apical-to-basal ventricular APD gradient. CONCLUSIONS: These data demonstrate that inhomogeneity of ventricular repolarization, as measured by DI, changes dynamically with CL shortening. Furthermore, the hypoxic ventricle does not attenuate DI after abrupt CL shortening and thereby lacks a physiological response expected to diminish vulnerability to arrhythmias.

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PubMed1995

Nonstationary vortexlike reentrant activity as a mechanism of polymorphic ventricular tachycardia in the isolated rabbit heart.

BACKGROUND: Ventricular tachycardia may result from vortexlike reentrant excitation of the myocardium. Our general hypothesis is that in the structurally normal heart, these arrhythmias are the result of one or two nonstationary three-dimensional electrical scroll waves activating the heart muscle at very high frequencies. METHODS AND RESULTS: We used a combination of high-resolution video imaging, electrocardiography, and image processing in the isolated rabbit heart, together with mathematical modeling. We characterized the dynamics of changes in transmembrane potential patterns on the epicardial surface of the ventricles using optical mapping. Image processing techniques were used to identify the surface manifestation of the reentrant organizing centers, and the location of these centers was used to determine the movement of the reentrant pathway. We also used numerical simulations incorporating Fitzhugh-Nagumo kinetics and realistic heart geometry to study how stationary and nonstationary scroll waves are manifest on the epicardial surface and in the simulated ECG. We present epicardial surface manifestations (reentrant spiral waves) and ECG patterns of nonstationary reentrant activity that are consistent with those generated by scroll waves established at the right and left ventricles. We identified the organizing centers of the reentrant circuits on the epicardial surface during polymorphic tachycardia, and these centers moved during the episodes. In addition, the arrhythmias that showed the greatest movement of the reentrant centers displayed the largest changes in QRS morphology. The numerical simulations showed that stationary scroll waves give rise to monomorphic ECG signals, but nonstationary meandering scroll waves give rise to undulating ECGs characteristic of torsade de pointes. CONCLUSIONS: Polymorphic ventricular tachycardia in the healthy, isolated rabbit heart is the result of either a single or paired ("figure-of-eight") nonstationary scroll waves. The extent of the scroll wave movement corresponds to the degree of polymorphism in the ECG. These results are consistent with our numerical simulations that showed monomorphic ECG patterns of activity for stationary scroll waves but polymorphic patterns for scroll waves that were nonstationary.

Animals↗
PubMed2002

Frequency-dependent breakdown of wave propagation into fibrillatory conduction across the pectinate muscle network in the isolated sheep right atrium.

Atrial fibrillation (AF) may result from stationary reentry in the left atrium (LA), with fibrillatory conduction toward the right atrium (RA). We hypothesize that periodic input to the RA at an exceedingly high frequency results in disorganized wave propagation, compatible with fibrillatory conduction. Simultaneous endocardial and epicardial optical mapping (di-4-ANEPPS) was performed in isolated, coronary-perfused sheep RA. Rhythmic pacing of Bachmann's bundle allowed well-controlled and realistic conditions for LA-driven RA. Pacing at increasingly higher frequencies (2.0 to 6.0 Hz) led to increasing delays in activation distal to major branching sites of the crista terminalis and pectinate bundles, culminating in spatially distributed intermittent blockade at or above approximately 6.5 Hz. At this "breakdown frequency," the direction of RA propagation became completely variable from beat to beat and thus transformed into fibrillatory conduction. Such frequency-dependent changes were independent of action potential duration. Rather, the spatial boundaries between proximal and distal frequencies correlated well with branch sites of the pectinate musculature. Thus, there exists a breakdown frequency in the sheep RA below which activity is periodic throughout the atrium and above which it is fibrillation-like. The data are consistent with the ideas that during AF, high-frequency activation initiated in the LA undergoes fibrillatory conduction toward the RA, and that sink-to-source effect at branch points of the crista terminalis and pectinate muscles is important in determining the complexity of the arrhythmia.

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PubMed2003

Transmural heterogeneity of calcium handling in canine.

Spatial heterogeneity of the action potential and its influence on arrhythmia vulnerability is known. However, heterogeneity of intracellular calcium handling and, in particular, its effect on the electrophysiological substrate is less clear. Using optical mapping techniques, calcium transients and action potentials were recorded simultaneously from ventricular sites across the transmural wall of the arterially perfused canine left ventricular wedge preparation during steady-state baseline pacing and rapid pacing. During baseline pacing, the decay of intracellular calcium to diastolic levels and calcium transient duration were slower (70%, P<0.005) and longer (20%, P<0.005), respectively, closer to the endocardial surface compared with the epicardial surface. Tissue samples isolated from the left ventricular wall demonstrate that sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression was significantly less in the subendocardial and midmyocardial layers compared with the subepicardial layer. In contrast, no significant difference in the transmural expression of Na+-Ca2+ exchanger was observed. During rapid pacing, calcium transient alternans and increased levels of diastolic intracellular calcium were significantly greater (P<0.01) closer to the endocardium (101%+/-62% and 41%+/-15%, respectively) compared with the epicardium (12%+/-7% and 12%+/-14%, respectively). In conclusion, cells closer to the endocardium exhibit a slower decay of intracellular calcium compared with cells near the epicardium, which may be due in part to reduced expression of SERCA2a. As a possible consequence, calcium transient alternans and increased diastolic levels of intracellular calcium may occur preferentially closer to the endocardial surface.

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PubMed2003

Impulse propagation in synthetic strands of neonatal cardiac myocytes with genetically reduced levels of connexin43.

Connexin43 (Cx43) is a major determinant of the electrical properties of the myocardium. Closure of gap junctions causes rapid slowing of propagation velocity (theta), but the precise effect of a reduction in Cx43 levels due to genetic manipulation has only partially been clarified. In this study, morphological and electrical properties of synthetic strands of cultured neonatal ventricular myocytes from Cx43+/+ (wild type, WT) and Cx+/- (heterozygote, HZ) mice were compared. Quantitative immunofluorescence analysis of Cx43 demonstrated a 43% reduction of Cx43 expression in the HZ versus WT mice. Cell dimensions, connectivity, and alignment were independent of genotype. Measurement of electrical properties by microelectrodes and optical mapping showed no differences in action potential amplitude or minimum diastolic potential between WT and HZ. However, maximal upstroke velocity of the transmembrane action potential, dV/dtmax, was increased and action potential duration was reduced in HZ versus WT. theta was similar in the two genotypes. Computer simulation of propagation and dV/dtmax showed a relatively small dependence of theta on gap junction coupling, thus explaining the lack of observed differences in theta between WT and HZ. Importantly, the simulations suggested that the difference in dV/dtmax is due to an upregulation of INa in HZ versus WT. Thus, heterozygote-null mutation of Cx43 produces a complex electrical phenotype in synthetic strands that is characterized by both changes in ion channel function and cell-to-cell coupling. The lack of changes in theta in this tissue is explained by the dominating role of myoplasmic resistance and the compensatory increase of dV/dtmax.

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PubMed2003

Nonlinear changes of transmembrane potential during electrical shocks: role of membrane electroporation.

Defibrillation shocks induce nonlinear changes of transmembrane potential (DeltaVm) that determine the outcome of defibrillation. As shown earlier, strong shocks applied during action potential plateau cause nonmonotonic negative DeltaVm, where an initial hyperpolarization is followed by Vm shift to a more positive level. The biphasic negative DeltaVm can be attributable to (1) an inward ionic current or (2) membrane electroporation. These hypotheses were tested in cell cultures by measuring the effects of ionic channel blockers on DeltaVm and measuring uptake of membrane-impermeable dye. Experiments were performed in cell strands (width approximately 0.8 mm) produced using a technique of patterned cell growth. Uniform-field shocks were applied during the action potential plateau, and DeltaVm was measured by optical mapping. Shock-induced negative DeltaVm exhibited a biphasic shape starting at a shock strength of approximately 15 V/cm when estimated peak DeltaV-m was approximately -180 mV; positive DeltaVm remained monophasic. Application of a series of shocks with a strength of 23+/-1 V/cm resulted in uptake of membrane-impermeable dye propidium iodide. Dye uptake was restricted to the anodal side of strands with the largest negative DeltaVm, indicating the occurrence of membrane electroporation at these locations. The occurrence of biphasic negative DeltaVm was also paralleled with after-shock elevation of diastolic Vm. Inhibition of I(f) and I(K1) currents that are active at large negative potentials by CsCl and BaCl2, respectively, did not affect DeltaVm, indicating that these currents were not responsible for biphasic DeltaVm. These results provide evidence that the biphasic shape of DeltaVm at sites of shock-induced hyperpolarization is caused by membrane electroporation.

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PubMed2004

Role of calcium cycling versus restitution in the mechanism of repolarization alternans.

T-wave alternans, a powerful marker of arrhythmic events, results from alternation in action potential duration (APD). The underlying cellular mechanism of APD alternans is unknown but has been attributed to either intracellular calcium (Ca2+) cycling or membrane ionic currents, manifested by a steep slope of cellular APD restitution. To address these mechanisms, high-resolution optical mapping techniques were used to measure action potentials and Ca2+ transients simultaneously from hundreds of epicardial sites in the guinea pig model of pacing-induced T-wave alternans (n=7). The pacing rates (ie, alternans threshold) at which T-wave (369+/-11 bpm), APD (369+/-21 bpm), and Ca2+ (371+/-29 bpm) alternans first appeared were comparable. Importantly, the site of origin of APD alternans and Ca2+ alternans consistently occurred together near the base of the left ventricle, not where APD restitution was steepest. In addition, APD and Ca2+ alternans were remarkably similar both spatially and temporally during discordant alternans. In conclusion, the mechanism underlying T-wave alternans in the intact heart is more closely associated with intracellular Ca2+ cycling rather than APD restitution.

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PubMed2004

Electrical propagation in synthetic ventricular myocyte strands from germline connexin43 knockout mice.

To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43-/- mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, gj, was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43+/+) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43+/- strands and was absent in Cx43-/- strands. Cx45 signal was reduced by 46% in Cx43+/- strands and to the limit of detection in Cx43-/- strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in 2% of myocytes. Intercellular conductance, gj, was reduced by 32% in Cx43+/- cell pairs and by 96% in Cx43-/- cell pairs. The symmetrical dependence of gj on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43-/- cells. Propagation in Cx43-/- strands was very slow (2.1 cm/s versus 52 cm/s in Cx43+/+) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands.

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PubMed2004

Epicardial fiber organization in swine right ventricle and its impact on propagation.

Fiber organization is important for myocardial excitation and contraction. It can be a major factor in arrhythmogenesis and current distribution during defibrillation shocks. In this study, we report the discovery of a previously undetected thin epicardial layer in swine right ventricle (RV) with distinctly different fiber orientation, which significantly affects epicardial propagation. Experiments were conducted in isolated coronary-perfused right ventricular free wall preparations (n=8) stained with the voltage-sensitive dye di-4-ANEPPS. Optical signals were recorded from the epicardium with a CCD video camera at 800 fps. Preparations were sectioned parallel to the epicardial surface with a resolution of 50 mum or better. To link the histological data with the observed activation patterns, resulting fiber angles were introduced into a 3D computer model to simulate the electrical activation and voltage-dependent optical signals. In all preparations, we detected a thin epicardial layer with almost no depth-dependent fiber rotation. The thickness of this layer (z(0)) varied from 110 to 930 microm. At the boundary of this layer, we observed an abrupt change in fiber angle by 64+/-13 degrees followed by a gradual fiber rotation in the underlying layers. In preparations with z(0) <700 microm, optical mapping during epicardial stimulation revealed unusual diamond- and rectangular-shaped activation fronts with two axes of fast conduction. Computer simulations accurately predicted the features of the experimentally recorded activation fronts. The free wall of swine RV has a thin epicardial layer with distinctly different fiber orientation, which can significantly affect propagation and give rise to unusually shaped activation fronts. This is important for understanding electrical propagation in the heart, and further refines the existing knowledge of myocardial fiber architecture.

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